Publication: Visualization of BRI1 and SERK3/BAK1 Nanoclusters in Arabidopsis Roots

J. Hohlbein and A.N. Kapanidis, Methods in Enzymology: Single-molecule Enzymology Part A & B, published online, 2016, [link] Monitoring conformational changes in DNA polymerases using single-molecule Förster resonance energy transfer (smFRET) has provided new tools for studying fidelity-related mechanisms that promote the rejection of incorrect nucleotides before DNA synthesis. In addition to the previously known open and the […]

S.J. Hutten, D.S. Hamers, M.A. an den Toorn, W. van Esse, A. Nolles, C.A. Bücherl, S.C. de Vries, J. Hohlbein, J.W. Borst, PLoS ONE 12(1): e0169905, 2017, [link]

Brassinosteroids (BRs) are plant hormones that are perceived at the plasma membrane (PM) by the ligand binding receptor BRASSINOSTEROID-INSENSITIVE1 (BRI1) and the co-receptor SOMATIC EMBRYOGENESIS RECEPTOR LIKE KINASE 3/BRI1 ASSOCIATED KINASE 1 (SERK3/BAK1). To visualize BRI1-GFP and SERK3/BAK1-mCherry in the plane of the PM, variable-angle epifluorescence microscopy (VAEM) was employed, which allows selective illumination of a thin surface layer. VAEM revealed an inhomogeneous distribution of BRI1-GFP and SERK3/BAK1-mCherry at the PM, which we attribute to the presence of distinct nanoclusters. Neither the BRI1 nor the SERK3/BAK1 nanocluster density is affected by depletion of endogenous ligands or application of exogenous ligands. To reveal interacting populations of receptor complexes, we utilized selective-surface observation—fluorescence lifetime imaging microscopy (SSO-FLIM) for the detection of Förster resonance energy transfer (FRET). Using this approach, we observed hetero-oligomerisation of BRI1 and SERK3 in the nanoclusters, which did not change upon depletion of endogenous ligand or signal activation. Upon ligand application, however, the number of BRI1-SERK3 /BAK1 hetero-oligomers was reduced, possibly due to endocytosis of active signalling units of BRI1-SERK3/BAK1 residing in the PM. We propose that formation of nanoclusters in the plant PM is subjected to biophysical restraints, while the stoichiometry of receptors inside these nanoclusters is variable and important for signal transduction.

Hutten_et_al2017

Publication: Fluorescence resonance energy transfer and protein-induced fluorescence enhancement as synergetic multi-scale molecular rulers

E. Ploetz, E. Lerner, F. Husada, M. Roelfs, S. Chung, J. Hohlbein, S. Weiss, T. Cordes, Scientific Reports, 6, 33257 , 2016, [link]

Advanced microscopy methods allow obtaining information on (dynamic) conformational changes in biomolecules via measuring a single molecular distance in the structure. It is, however, extremely challenging to capture the full depth of a three-dimensional biochemical state, binding-related structural changes or conformational cross-talk in multi-protein complexes using one-dimensional assays. In this paper we address this fundamental problem by extending the standard molecular ruler based on Förster resonance energy transfer (FRET) into a two-dimensional assay via its combination with protein-induced fluorescence enhancement (PIFE). We show that donor brightness (via PIFE) and energy transfer efficiency (via FRET) can simultaneously report on e.g., the conformational state of dsDNA following its interaction with unlabelled proteins (BamHI, EcoRV, T7 DNA polymerase gp5/trx). The PIFE-FRET assay uses established labelling protocols and single molecule fluorescence detection schemes (alternating-laser excitation, ALEX). Besides quantitative studies of PIFE and FRET ruler characteristics, we outline possible applications of ALEX-based PIFE-FRET for single-molecule studies with diffusing and immobilized molecules. Finally, we study transcription initiation and scrunching of E. coli RNA-polymerase with PIFE-FRET and provide direct evidence for the physical presence and vicinity of the polymerase that causes structural changes and scrunching of the transcriptional DNA bubble.

2016_Ploetz

Publication: A Quantitative Theoretical Framework For PIFE-FRET

E. Lerner, E. Ploetz, J. Hohlbein, T. Cordes, S. Weiss, The Journal of Physical Chemistry B, 120, 6401–6410, 2016, [link]

Single molecule protein induced fluorescence enhancement (PIFE) serves as a molecular ruler at molecular distances inaccessible to other spectroscopic rulers such as Förster-type resonance energy transfer (FRET) or photo-induced electron transfer. In order to provide two simultaneous measurements of two distances on different molecular length scales for the analysis of macromolecular complexes, we and others recently combined measurements of PIFE and FRET (PIFE-FRET) on the single molecule level. PIFE relies on steric hindrance of the fluorophore Cy3, which is covalently attached to a biomolecule of interest, to rotate out of an excited-state trans isomer to the cis isomer through a 90 deg intermediate. In this work, we provide a theoretical framework that accounts for relevant photophysical and kinetic parameters of PIFE-FRET, show how this framework allows the extraction of the fold-decrease in isomerization mobility from experimental data and how these results provide information on changes in the accessible volume of Cy3. The utility of this model is then demonstrated for experimental results on PIFE-FRET measurement of different protein-DNA interactions. The proposed model and extracted parameters could serve as a benchmark to allow quantitative comparison of PIFE effects in different biological systems.

 

EitanPife

Publication: Single molecule 3D orientation in Time and Space: A 6D dynamic study on fluorescent labeled lipid membranes.

R. Börner, N. Ehrlich, J. Hohlbein, C.G. Hübner, Journal of Fluorescence, 26, 963-975, 2016 [link]

Interactions between single molecules profoundly depend on their mutual three-dimensional orientation to each other. Recently, we demonstrated a technique that allows the orientation determination of single dipole emitters using a polarization-resolved distribution of fluorescence into several detection channels. As tCapture2he method is based on the detection of single photons, it additionally allows for performing fluorescence correlation spectroscopy (FCS) as well as dynamical anisotropy measurements thereby providing access to fast orientational dynamics down to the nanosecond time scale. The 3D orientation is particularly interesting in non-isotropic environments such as lipid membranes, which are of great importance in biology. We used giant unilamellar vesicles (GUVs) labeled with fluorescent dyes down to a single molecule concentration as a model system for both, assessing the robustness of the orientation determination at different timescales and quantifying the associated errors. The vesicles provide a well-defined spherical surface, thus, the in cooperation of lipid dyes (DiO) represents a a wide range of dipole orientations. To complement our experimental data, we performed Monte Carlo simulations of the rotational dynamics of dipoles incorporated into lipid membranes. Our study offers a comprehensive view on the dye orientation behavior in a lipid membrane with high spatiotemporal resolution representing a six-dimensional fluorescence detection approach. 

Publication: Complex coacervate core micelles with spectroscopic labels for diffusometric probing of biopolymer networks

N. Bourouina, D. de Kort, F. Hoeben, H. Janssen, H. Van As, J. Hohlbein, J. van Duynhoven, J.M. Kleijn, Langmuir, 31, 12635-12643, 2015, [link]

We present the design, TableOfContentpreparation and characterization of two types of complex coacervate core micelles (C3Ms) with cross-linked cores and spectroscopic labels, and demonstrate their use as diffusional probes to investigate the microstructure of percolating biopolymer networks. The first type consists of poly(allylamine hydrochloride) (PAH) and poly(ethylene oxide)-poly(methacrylic acid) (PEO-b-PMAA), labeled with ATTO 488 fluorescent dyes. We show that the size of these probes can be tuned by choosing the length of the PEO-PMAA chains. ATTO 488-labeled PEO113-PMAA15 micelles are very bright with 18 dye molecules incorporated into their cores. The second type is a 19F-labeled micelle, for which we used PAH and a 19F-labeled diblock copolymer tailor-made from poly(ethylene oxide) poly(acrylic acid) (mPEO79-b-PAA14). These micelles contain approximately 4 wt% of 19F and can be detected by 19F NMR. The 19F labels are placed at the end of a small spacer to allow for the necessary rotational mobility. We used these ATTO- and 19F-labeled micelles to probe the microstructures of a transient gel (xanthan gum) and a cross-linked, heterogeneous gel (kappa-carrageenan). For the transient gel, sensitive optical diffusometry methods, including fluorescence correlation spectroscopy, fluorescence recovery after photobleaching and super-resolution single nanoparticle tracking allowed us to measure the diffusion coefficient in networks with increasing density. From these measurements, we determined the diameters of the constituent xanthan fibers. In the heterogeneous kappa-carrageenan gels, bi-modal nanoparticle diffusion was observed, which is a signpost of microstructural heterogeneity of the network.

Publication: New technologies for DNA analysis – a review of the READNA Project

S. McGinn, D. Bauer, T. Brefort, L. Dong, A. El-Sagheer, A. Elsharawy, G. Evans, E. Falk-Sörqvist, M. Forster, S. Fredriksson, P. Freeman, C. Freitag, J. Fritzsche, S. Gibson, M. Gullberg, M. Gut, S. Heath, I. Heath-Brun, A.J. Heron, J. Hohlbein, R. Ke, O. Lancaster, L. Le Reste, G. Maglia, R. Marie, F. Mauger, F. Mertes, M. Mignardi, L. Moens, J. Oostmeijer, R. Out, J. Nyvold Pedersen, F. Persson, V. Picaud, D. Rotem, N. Schracke, J. Sengenes, P.F. Stähler, B. Stade, D. Stoddart, X. Teng, C.D. Veal, N. Zahra, H. Bayley, M. Beier, T. Brown, C. Dekker, B. Ekström, H. Flyvbjerg, A. Franke, S. Guenther, A.N. Kapanidis, J. Kaye, A. Kristensen, H. Lehrach, J. Mangion, S. Sauer, E. Schyns, J. Tost, J.M.L.M. van Helvoort, P.J. van der Zaag, J. O. Tegenfeldt, A.J. Brookes, K.Mir, M. Nilsson, S. Willcocks, I.G. Gut, New Biotechnology, 33, 310-330, 2016, [link]

The REvolutionary Approaches and Devices for Nucleic Acid analysis (READNA) project received funding from the European Commission for 4 1/2 years. The objectives of the project revolved around technological developments in nucleic acid analysis. The project partners have discovered, created and developed a huge body of insights into nucleic acid analysis, ranging from improvements and implementation of current technologies to the most promising sequencing technologies that constitute a 3rd and 4th generation of sequencing methods with nanopores and in situ sequencing, respectively.

Publication: Camera-based single-molecule FRET detection with improved time resolution

S. Farooq and J. Hohlbein, Physical Chemistry Chemical Physics, 17, 27862, 2015, [link], open access

The achievable time resolution of camera-based single-molecule detection is often limited by the frame rate of the camera. Especially in experiments utilizing single-molecule Förster resonance energy transfer (smFRET) to probe conformational dynamics of biomolecules, increasing the frame rate by either pixel-binning or cropping the field of view decreases the number of molecules that can be monitored simultaneously. Here, we present a generalised excitation scheme termed stroboscopic alternating-laser excitation (sALEX) that significantly improves the time resolution without sacrificing highly parallelised detection in total internal reflection fluorescence (TIRF) microscopy. In addition, we adapt a technique known from diffusion-based confocal microscopy to analyse the complex shape of FRET efficiency histograms. We apply both sALEX and dynamic probability distribution analysis (dPDA) to resolve conformational dynamics of interconverting DNA hairpins in the millisecond time range.